The long-term goal of the proposed research is to characterize and understand the modifications in axonal transport in Alzheimer's disease (AD). Although it was suggested that an abnormal axonal transport could contribute to AD pathology, no systematic study was conducted. Here, we aim to cover this gap. We begin by asking how the amyloid-? precursor protein (APP), a type I transmembrane protein that is at the core of the pathogenic process in AD, is transported to the site(s) of its function within neuronal processes, in normal and in disease-related conditions. This study should have a strong impact on human health. APP, the precursor of the amyloid-? peptide that forms the senile plaques in AD, is proteolytically cleaved, by the action of secretases, into soluble, N-terminal (sAPP) and C-terminal (CTF) polypeptides. We recently showed that, in neurons, proteolytic processing of APP into fragments largely occurs prior to sorting into cargo vesicles, and that the different APP-derived polypeptides are transported independently (by distinct vesicle populations) to different destinations (Muresan et al., 2009. J. Neuroscience, 29: 3565-3578). To begin characterizing APP transport we will start with characterizing the transport of full-length APP and of the APP-derived N- and C-terminal fragments. To achieve this goal, we will generate knock-in mice that express APP, tagged with different reporters at its termini, which will allow separate tracking of the APP's N- and C-terminal fragments. Most importantly, because the dual-tagged APP will be knocked into the endogenous APP locus by homologous recombination (to maintain the endogenous regulatory elements for APP expression), the dual-tagged APP will be expressed at endogenous levels. We propose two Specific Aims. In Specific Aim I, we will generate the knock-in mice that will allow us to analyze the processing, transport, and localization of APP, and investigate the spectrum of proteins that interact with the APP's N- and C-terminal fragments. With in vivo motility assay, in Specific Aim II we will analyze the transport of APP-derived polypeptides in the neurons from the knock-in mice expressing CFP-APP-YFP (with CFP and YFP tagging the APP's N- and C- termini). An additional knock-in mouse expressing FLAG-APP-myc will be useful to determine with biochemical and subcellular fractionation methods the organelles where the APP fragments target, and the different sets of proteins that cotransport or interact with APP polypeptides. Finally, this study will emphasize the relation between the transport, processing, and function(s) of APP, and will increase the understanding of APP biology in the context of AD.